PROJECT SUMMARY Francisella tularensis (Ft) is a potential agent of bioterror, facultative intracellular pathogen and the causative agent of tularemia. Based on several of its attributes, Ft has been named by the CDC as a Tier 1 agent. While several advances have been made in the last ~15 years regarding the cell biology of this pathogen, its method of virulence gene regulation remains incompletely understood. To survive and thrive in often hostile environments, a bacterium has to monitor its surroundings and adjust its gene expression and physiology accordingly. This is especially important for pathogenic bacteria that continuously interact with the host during an infection. Gene regulation is traditionally mediated by protein regulatory factors, but Ft contains few protein regulatory factors and is devoid of classically arranged two-component regulatory systems, yet mediates complex host-pathogen processes such as suppression of the immune response early after host contact and escape from the Francisella-containing vacuole into the cytosol. A limited number of orphan two-component members exist including the response regulator PmrA. PmrA is a key factor in Francisella pathogenesis, as it has been demonstrated to regulate Ft biofilm formation, intramacrophage survival and animal virulence. Small RNAs (sRNAs) are also increasingly associated with the regulation of virulence in pathogens, and we have identified 661 Ft sRNAs, including the subset regulated by PmrA (n=81). The overall goal of this research is to understand the transcriptional/post-transcriptional control of virulence in this important pathogen. The overall objective is to identify virulence-associated sRNAs (PmrA-regulated as well as those differentially expressed in macrophages). The central hypothesis is that Ft sRNAs are influenced by PmrA and the macrophage environment to regulate virulence. The achievement of our goals will uncover new targets for Ft vaccines or for therapeutics to counteract this dangerous pathogen.